Project Summary/Abstract Malaria continues to cause substantial morbidity and mortality throughout the developing world. The development of resistance to newly deployed drugs as well as the lack of an effective vaccine indicate that this disease will continue to plague mankind for the foreseeable future. It is caused by infection with mosquito-borne parasites of the genus Plasmodium, with the species P. falciparum being responsible for the most severe form of the disease. These parasites utilize a process called antigenic variation to avoid the adaptive immune response of their host and thus maintain long-term, chronic infections that can last over a year. Such lengthy infections provide the parasites with ample opportunities to be transmitted to additional human hosts through blood feeding by Anopheline mosquitoes. Infection of mosquitoes requires the differentiation of parasites into male and female gametocytes, a process that involves a cascade of gene expression that initiates with the expression of a master regulator of sexual differentiation called PfAP2-G. Both antigenic variation and sexual differentiation result from highly regulated mechanisms of gene activation and silencing that are controlled epigenetically through changes in histone methylation patterns at key positions in the genome. These changes in gene expression have been presumed to happen stochastically, occurring at a rate that has evolved to provide the parasites with the best opportunity for successful transmission between hosts. In this proposal, we explore the possibility that rather than occurring stochastically, changes in gene expression that lead to both antigenic variation and sexual differentiation occur in response to changes in the parasite's environment. For antigenic variation, we hypothesize that parasites can sense rising antibody titers that recognize var gene-encoded RBC surface antigens, triggering switching of expression to alternative members of the var gene family. Similarly, we hypothesize that parasites can sense when they invade erythrocyte precursor cells and respond by committing to sexual differentiation through the activation of the gene encoding PfAP2-G. This results in preferential development of gametocytes in reticulocytes and in the bone marrow, as has been observed in vivo. We have identified a metabolic pathway that links both of these responses to changes in intracellular stores of the universal methyl donor, S-adenosylmethionine. Through simple manipulations of the availability of key nutrients that feed into this pathway, we can trigger either var gene switching or sexual commitment in cultured parasites. The specific aims of the project are designed to investigate how changes in nutrient availability are translated into changes in intracellular S-adenosylmethionine, how these changes affect histone methylation patterns at var genes and pfap2-g, and to identify the physiological conditions that trigger var gene switching or sexual commitment in vivo. The results will alter the way we understand how malaria parasites sense and respond to changes in their environment.